Graphics Gallery

Gale Rhodes
Chemistry Department
University of Southern Maine

Revised 2006/11/07

Learn how to use Swiss-PdbViewer. Work through sections 1-4 of the Swiss-PdbViewer Tutorial.

Topic: Lipids and Membranes

Thanks for Eric Martz for preparing the Mebrane Lipids and Ionophores files in PDB format.
They are part of a RasMol animation of lipids and bilayers, available at the RasMol Home Page.

For a full description of the bilayer models provided and discussed on this page, see "Molecular dynamics simulation of a bilayer of 200 lipids in the gel and in the liquid-crystal phases", H. Heller, M. Schaeffer, and K. Schulten, J. Phys. Chem, 97, 8343-60 (1993).

 

Examples:

Membrane Lipids

Cholesterol, Phosphatidyl Choline, and Lipid Bilayers (Deep View project file)

NOTES, Layer 1, cholesterol

  • Cholesterol is amphipathic, having both polar and nonpolar regions. What functional group constitutes the polar region.
  • Cholesterol is a normal component of cell membranes. How would it be oriented in a lipid bilayer?
  • Cholelsterol stiffens the outer parts of the phospholipid tails, thus making the membrane less fluid.
  • Counterintuitively, cholesterol also acts as an impurity, lowering the "freezing" temperature of the membrane -- the temperature at which the membrane undergoes a transition from the fluid phase to the gel phase. So it lowers membrane fluidity, but widens the temperature range over which the membrane remains fluid.

NOTES, Layer 2, phosphatidyl choline (PC)

  • Note the amphipathic nature of PC.
  • Identify the fatty acids in these PC molecules. Each molecule has two different fatty acids esterified to it.
  • Display cholesterol and PC together. Approximately how would their polar heads align if they were neighbors in a membrane?

NOTES, Layer 3, 40 PC molecules in a lipid bilayer, with water molecules on one side.

  • Compute hydrogen bonds and look for examples of H-bonds between water molecules and PC head groups. Does the absence of H-bonds involving the positively charged triethyammonium groups surprise you? Explain.
  • Note that all fatty-acid tails are shown with the same conformation. This is an idealized model. A larger model like it was used as a starting point for molecular dynamics simulations of motion in lipid bilayers. To see the resulting nonidealized model after 100 picoseconds of computer-simulated motion, click here (NOTE: large file!). This will add a fourth layer, called Fluid, to the display. This model is probably much more like a real bilayer.
  • Compare the idealized and "real" models as to 1) the variety of hydrocarbon conformations, 2) thickness of the bilayers. Why is the dynamics model thinner?
  • For a smaller non-idealized bilayer model, see the gramicidin model next.

Phospholipases

A Competitive Inhibitor of Phospholipase A2

Click the link above to download a Deep View file derived from PDB file 1POE.pdb (click this filename to learn more about this protein directly from its PDB entry).

Phospholipase A2 catalyzes hydrolysis of fatty-acid links in phospholipids like phosphatidylethanolamine (PE), releasing fatty acids from the 2-position of glycerol. Phospholipase model 1POE includes a nonprotein ligand (designated GEL) which is an unreactive analog of PE. This ligand binds tightly to the active site of the enzyme, excluding its normal substrate, and thus reducing the rate of ester hydrolysis. GEL is therefor called a competitive inhibitor of phospholipase A2 -- it inhibits the enzyme by competing with the substrate, the substance on which the enzyme normally acts.

When you open this file, you will find GEL displayed with a van der Waals surface, its neighbors (to 4 angstroms) displayed as stick models, and the rest of the protein in ribbon.

NOTES

  • Study the structure of GEL (dotted surface), and determine in what ways it is like, and unlike, PE.
  • Display hydrogen bonds between protein and ligand to see how the polar groups of the ligand are stabilized by the protein.
  • A PREVIEW: HOW ENZYMES WORK: Write the mechanism for base-promoted hydrolysis of an ester. The product of the rate-determining step is a tetrahedral intermediate resulting from addition of hydroxide ion to the the ester carbonyl. Because this intermediate is near the transition state on the progress-of-reaction curve for this process, the transition state must therefore be structurally very similar to this tetrahedral intermediate.
  • Compare the phosphodiester group at C2 of the glycerol to the tetrahedral intermediate in ester hydrolysis -- they are very similar in geometry and electronic structure. Thus, to this enzyme, GEL looks much more like the transition state in ester hydrolysis than like its reactant (ester) or product (carboxylic acid). If an enzyme binds strongly to, and thus stabilizes, the transition state of the reaction it catalyzes, then it will lower the energy required to reach the transition state and make the reaction faster. It appears that stabilization of the transition state is at least one aspect of how phospholipases catalyze ester hydrolysis.
  • Inhibitors like GEL are called transition-state analogues. Scientists can often use knowledge of enzyme reaction mechanisms to design such inhibitors with the aim of selectively blocking enzyme action. Some important drugs are transition-state analogues for vital enzymes in pathogens.
  • Protein-digesting enzymes know as serine proteases hydrolyze peptide bonds by a mechanism similar to that of ester hydrolysis by phospholipases. Look up the mechanism of action of serine proteases in your biochemistry textbook. You will probably find mention of an oxyanion hole at the enzyme's active site. Can you find something similar to an oxyanion hole in phospholipase A2? Look at how the negative charges on the glycerol C-2 phosphate are stabilized.

To see this enzyme in the presence and absence of GEL, click HERE.

  • In layer 1POE, select GEL and then select and display its neighbors to 4.5 angstroms. Select: Extend to other layers to make the same selection in layer 1POD, which lacks GEL. Move to layer 1 POD and press return to display only the selected residues.
  • Move back and forth between 1 POD and 1 POE (control-tab) to see conformational differences between the two models. These differences accomodate the substrate and move transition-state stabilizing groups into position.

Ionophores

Gramicidin in Lipid Bilayer

NOTES

  • Gramicidin is an ionophore, or ion carrier. It make membranes permeable to water and protons, but its permeability is blocked by Ca2+ ions.
  • Notice that the lipid side chains from the two monolayers intermingle with each other. This model was produced by computer simulation of the motion of all molecules, starting from an idealized model like the one described above. This model is probably much like a real bilayer.
  • Notice the water molecules in the gramicidin channel.
  • Rotate the model to look at it end-on. Only half the bilayer needed to enclose the gramicidin molecule is shown in the model.
  • Notice that the ionophore is polar inside and hydrophobic outside -- just the opposite of water-soluble proteins. What polar groups make up the interior? How does the distribution of polar and nonpolar groups compare with that of a water-soluble protein? 

Valinomycin

NOTES (read all before clicking the link at the end):

  • Not a channel former like gramicidin, valinomycin wraps potassium ions in a 36-member chain of amino- and hydroxy-acid residues, giving it a hydrophobic covering in which it can traverse the nonpolar regions of a lipid bilayer.
  • To view the molecule with Deep View, click here.

Integral Membrane Proteins

Bacteriorhodopsin (PDB 1C3W)

NOTES

  • Bacteriorhodopsin is a light-driven proton pump from a halophilic bacterium. Its action produces a pH gradient across the cell membrane. This gradient is the primary energy source for all of the bacterium's cellular metabolism, including the production of ATP for driving chemical processes like biosynthesis. The mechanism of proton pumping is an active area of research. Bacteriorhodopsin is the best understood transmembrane pump, but there are still many questions about exactly how it works.
  • "The function of bacteriorhodopsin has been studied extensively by a variety of structural, genetic, and spectroscopic methods and by molecular dynamics calculations. The primary event, absorption of a photon, causes isomerization of the retinal from the all-trans to the 13-cis configuration. A series of intermediate events follows, including deprotonation of the Schiff base and proton transfer to Asp85. Another proton is subsequently released, the Schiff base is reprotonated from Asp96, and a proton is taken up from the cytoplasmic side. Thermal reisomerization of the retinal to the ground state completes the photocycle." (from X-ray Structure of Bacteriorhodopsin at 2.5 Angstroms from Microcrystals Grown in Lipidic Cubic Phases, Eva Pebay-Peyroula, Gabriele Rummel, Jurg P. Rosenbusch, Ehud M. Landau Science, Volume 277, Number 5332, Issue of 12 Sep 1997, pp. 1676-1681.)

    All-trans retinal
  • Download a bacteriorhodopsin model by clicking on the PDB file name above. Display the backbone as ribbon and color by secondary structure. What is the main type of secondary structure in this molecule? Why is this type of structure often found in transmembrane regions of proteins?
  • Display backbone and color by secondary structure succession. Trace the chain from N- to C-terminus. Notice how this color scheme makes it easy to follow the chain through the structure, even if there are breaks in the sequence. Locate one such break and use labeling to identify the residue numbers on each side of the break. Why do you think some residues are missing from the model? (Hint: the structure was determined by x-ray crystallography.)
  • Color backbone dark gray (so it id very faint against the blace background), and color sidechains by type. By looking at the distribution of sidechain types (charged, polar, nonpolar) on the molecular surface, can you tell what parts of this molecule are buried in the cell membrane and what parts are exposed at the membrane faces?
  • Notice a blue sidechain in the interior. What amino acid is it? Why is it unusual to find this amino acid buried within a protein? This sidechain is not its usual ionic self, however. It is covalently attached to retinal, the free form of which is an aldehyde. What type of chemical attachment is this, between an amine and an aldehyde?
  • Examine the contacts between retinal and the protein interior (Use Select: Neighbors... to limit the display). What types of interactions do you find? The fundamental event in proton pumping is the absorption of a photon of light by retinal, resulting in isomerization of one of its trans-double bonds to the cis-isomer. The change in shape induces conformational changes in the protein, which are thought to be essential to translocation of a proton from one end of the molecule to the other, and hence across the membrane. Would you describe the retinal pocket as loose- or tight-fitting? Which would be conducive to coupling isomerization of the retinal to conformational change in the protein?

Bacteriorhodopsin "In Action"

Click HERE to download a DeepView project file that depicts light-driven proton pumping by bacteriorhodopsin. Before loading this file, make sure that DeepView loading preferences (Prefs:Loading Protein) are set to load and show water molecules.

Crystallographers have determined the structures of many site-directed mutants of this protein, including some whose spectral properties suggest that the mutations have trapped them in conformations that represent intermediates in the pumping process. I have assembled a set of these models to depict proton pumping. The models appear in the project file in the order thought to represent the stages of pumping. The protons are depicted by water models with dotted surfaces. If you blink through this series, following a dotted ball of a specific color, you will see it associate with the side-chain groups that have been proposed as the proton-carrier sites. I emphasize that this is not an animation of proton pumping. It is simple a series of crystallographic models upon which one can illustrate the action of proposed proton carrying groups.

When the retinal model goes from purple to dark blue, it is depicting the trans-to-cis isomerization that occurs on light absorption. The models shown between that and the next light-absorption event represent pumping stages, including at one point the re-isomerization of retinal to the trans- form, which probably occurs spontaneously. At the cis-to-trans reisomerization, the retinal model goes from yellow to light gray.

The first layer (named chain) of this file is a full model, while all other layers are partial. For the most dramatic depiction, use the Wind:Layers Infos command to control the layers. In the cyc column, click the first layer's entry repeatedly to turn it to a "+", which keeps in it view while your blink through all other layers (they should have checkmarks in the cyc column). In the chain layer, turn off display of all residues, leaving only the ribbon. Use Prefs:Ribbons to display the ribbon as a single strand. Now blink (hold down ctrl and press tab repeatedly) to see the action. For the fastest pumping, hold down ctrl and then just hold down tab.

OmpF Porin from E. coli (Deep View project file)

NOTES

  • Porins are channel forming proteins in the outer membranes of gram-negative bacteria, mitochondria, and choloroplasts. They permit entry of solutes smaller than 600kD, such as nutrients. What is the signficance of the presence of porins in these particular membranes, but not in the cytoplasmic membranes of eucaryotes?
  • The initial display in Deep View is ribbon backbone. Beta strands are white, alpha helices are red, and loops are a different color for each monomer of this oligomeric molecule. Also on display are some detergent molecules that were part of the crystallization medium. They are the same color as the chain with which they are associated. What is the subunit composition of this porin?
  • What is its primary type of secondary structure? Color the ribbon by secondary structure succession and trace the chain from N-terminus (near the strand of darkest blue) to C-terminus (reddest strand).
  • Remove ribbons, and display the backbone of chain A. Color backbone (only) CPK. Compute H-bonds and examine hydrogen bonding in the outer shell of the structure. What are beta barrels and alpha helices the two most common kinds of secondary structure found in transmembrane regions of proteins?
  • Color backbone dark gray (so it id very faint against the blace background), and color sidechains by type (they are already colored this way if you have followed these instructions carefully). Study the types of residues that cover the porin surface. By looking at side-chain types (charged, polar, nonpolar) can you tell what parts of the outer surface are buried in the membrane and what parts are exposed at the membrane faces?
  • The transmembrane channel is constricted by what type of structure? Look straight down into the pore. Use a 10-angstrom slab to travel through the pore, as follows: Turn on slab, then hold down <command> while you translate the molecule. In effect this action slides the molecule along the z-axis through the display slab, enabling you to see a moving thin section of the structure. What are the predominant types of residues that line the constricted part of the channel?
  • Display all chains and look down the transmembrane direction into the subunit interface. Then select and display only those residues that are within 5 angstroms of the subunit interface (Select: Groups Close to Another Chain...). This limits the display to the residues that make interchain contacts. Now use the slab to move through this interface. What types of interactions hold the oligomers together?
  • OmpF porin is weakly cation selective in permitting solutes to cross the membrane. A similar channal molecule, PhoE porin, is weakly anion selective. Obtain the original PDB for both porins (2OMF and 1PHO) and compare them. Your biochemistry text might have additional information about porins. If not, Google "porin".

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